Use of xianglongsin in the preparation of a medicine for preventing and treating porcine reproductive and respiratory syndrome

Abstract

The application is a divisional application of application number 202210068504.1, application date January 20, 2022, and invention name 'Use of Toosendanin in Preparation of Medicine for Prevention and Treatment of Porcine Viral Infectious Disease'. The present application belongs to the technical field of veterinary medicine, and specifically relates to the use of toosendanin in the preparation of medicine for prevention and treatment of porcine reproductive and respiratory syndrome. The present application provides a new antiviral use of toosendanin. According to the research of various methods, it is proved that toosendanin can significantly inhibit the proliferation of reproductive and respiratory syndrome virus at a micromolar concentration level, and can achieve the effect of preventing and treating porcine reproductive and respiratory syndrome. Moreover, toosendanin is widely sourced, low in cost, high in safety in clinical application, and very suitable for large-scale industrial production and use.

Description

[0001] This application is a divisional application of application No. 202210068504.1, titled "Use of Toosendanin in Preparation of Medicine for Preventing and Treating Porcine Viral Infectious Disease", filed on January 20, 2022. TECHNICAL FIELD

[0002] The present application belongs to the technical field of veterinary medicine. More specifically, it relates to the use of toosendanin in the preparation of medicine for preventing and treating porcine reproductive and respiratory syndrome. BACKGROUND

[0003] Porcine reproductive and respiratory syndrome (PRRS) is an infectious disease characterized by respiratory symptoms in pigs and reproductive disorders in sows, caused by porcine reproductive and respiratory syndrome virus (PRRSV). Due to the symptoms of blue ears in infected pigs, it is commonly known as "porcine blue ear disease". The main clinical symptoms are depression, lethargy, anorexia, cyanosis of the skin and ears, and high fever (40-42℃) in infected pigs. Most infected pigs show typical respiratory symptoms such as rhinorrhea, sneezing, and dyspnea, and sows show reproductive symptoms such as abortion and stillbirth. In 2006, highly pathogenic porcine reproductive and respiratory syndrome (HP-PRRS) broke out, characterized by high fever, high morbidity, and high mortality. This virus can cause 80-100% of pigs to be infected and 20-100% of pigs to die, causing significant losses to the pig industry in China.

[0004] From 2014 to 2016, Wang Kewen and Yang Hanchun of China Agricultural University conducted RT-PCR detection of PRRSV on 224 clinical samples collected from 56 pig farms in 15 provinces. The results showed that 163 samples were detected with virus, with an infection rate of 72%, of which 76% were infected with highly pathogenic PRRSV (Wang Kewen (2016). Evaluation and analysis of clinical immunization effect of attenuated live vaccine of highly pathogenic porcine reproductive and respiratory syndrome virus TJM-F92 strain, Doctoral Dissertation of China Agricultural University). This result reflects that PRRS has been widely present in pig farms and is one of the most harmful diseases to the pig industry at present, and effective prevention and control of PRRS is urgent. On the other hand, there are many subtypes of PRRSV, and the immune effect of the vaccine is not ideal.

[0005] In recent years, there are more and more reports on the research of anti-PRRSV drugs, but most of the related researches are at a relatively primary level, and the effective concentration of the active compounds is high, and the mechanism of action is not clear, which is difficult to meet the four basic requirements of "safety, effectiveness, stability and controllability" as a drug, and the drug-making property is poor. Therefore, the research and development of anti-viral drugs for porcine reproductive and respiratory syndrome is very urgent. SUMMARY

[0006] The technical problem solved by the present application is to provide a use of toosendanin in the preparation of a drug for preventing and treating porcine reproductive and respiratory syndrome, aiming at the deficiency that there is no drug for treating PRRS in veterinary clinics.

[0007] The above object of the present application is achieved by the following technical solutions.

[0008] Toosendanin (TSN) is a kind of tetracyclic triterpenoid compound extracted from the traditional anthelmintic Chinese medicine Melia Toosendain Sieb. et Zucc or Melia azedarach L. or the fruit of Melia Toosendain Sieb. et Zucc, which has the structure of formula (I). Toosendanin is mainly used for agricultural pest control, and has been used as an anthelmintic drug in medical clinics in China for more than 50 years. In addition, toosendanin also has the biological activities of resisting botulism, blocking neuromuscular junction and affecting central nervous system, efferent nerve and respiratory system; in recent years, it has been found that toosendanin also has certain anti-tumor activity (Luo Weiw, Lu Jinjian, et al. (2016), Research Progress of Pharmacological Effects and Mechanisms of Toosendanin, Chinese Traditional and Herbal Drugs, 32(04): 161-164.).

[0009]

[0010] The inventors have found, through a large amount of creative labor, that toosendanin has a significant antiviral effect, especially against African swine fever virus, porcine reproductive and respiratory syndrome virus and porcine epidemic diarrhea virus.

[0011] Therefore, the present application claims the use of toosendanin in the preparation of a drug for preventing and treating porcine viral infectious diseases.

[0012] Further, the porcine viral infectious disease is African swine fever, porcine reproductive and respiratory syndrome or porcine epidemic diarrhea.

[0013] Still further, the African swine fever is caused by infection of African swine fever virus.

[0014] Preferably, for the African swine fever virus, the effective virus inhibition concentration of toosendanin ranges from 0.1 to 3.0 μM.

[0015] Further, the porcine reproductive and respiratory syndrome is caused by infection of porcine reproductive and respiratory syndrome virus.

[0016] Preferably, the effective virus inhibition concentration of the meliatoxin is (0.1-1.6) μM against the porcine reproductive and respiratory syndrome virus.

[0017] Further, the porcine epidemic diarrhea is caused by infection of porcine epidemic diarrhea virus.

[0018] Preferably, the effective virus inhibition concentration of the meliatoxin is (2.0-8.0) μM against the porcine epidemic diarrhea virus.

[0019] Further, the meliatoxin is extracted from the root bark and / or the bark of Melia toosendan and / or the fruit of the plant, i.e., the melia fruit.

[0020] Still further, the meliatoxin can be prepared by the following method:

[0021] The root bark and / or the bark of Melia toosendan is crushed and sieved, and 80-100% ethanol solution is heated in water bath at 45-60℃ for 1-3 times, each time for 1-3 hours, and the filtrates are combined and concentrated to obtain a crude extract; the crude extract is extracted with petroleum ether, ethyl acetate and n-butanol in sequence, and the ethyl acetate extraction part is concentrated under reduced pressure; the obtained concentrated paste is subjected to silica gel column chromatography, eluted with 1% methanol-chloroform, 2% methanol-chloroform and 5% methanol-chloroform in sequence, and the eluate containing meliatoxin is collected and concentrated under reduced pressure, and then subjected to silica gel column chromatography again and eluted with 1% methanol-chloroform; the eluate is subjected to dextran gel LH-20 column and eluted with 50% methanol-water to obtain purified meliatoxin.

[0022] The meliatoxin can also be purchased directly.

[0023] Further, the medicine can be injection preparation, oral preparation, atomization inhalation preparation or transdermal preparation.

[0024] The present application has the following beneficial effects:

[0025] The present application provides a new antiviral use of meliatoxin, and studies by various methods prove that meliatoxin can significantly inhibit the proliferation of porcine reproductive and respiratory syndrome virus at micromolar concentration level, and can achieve the effect of preventing and treating porcine reproductive and respiratory syndrome. Moreover, meliatoxin is widely available and low in cost, and is safe in clinical application, and is very suitable for large-scale industrial production and use. BRIEF DESCRIPTION OF DRAWINGS

[0026] Figure 1 Statistical diagram of the inhibition of different concentrations of meliatoxin on the proliferation of ASFV DNA in PAMs cells in Example 1;

[0027] Figure 2Figure 2. Inhibition of ASFV RNA transcription in PAMs cells by different concentrations of toosendanin. The data were analyzed by one-way ANOVA followed by Dunnett's test.

[0028] Figure 3 Figure 3. Inhibition of ASFV P30 protein synthesis in PAMs cells by different concentrations of toosendanin. The data were analyzed by one-way ANOVA followed by Dunnett's test.

[0029] Figure 4 Figure 4. Inhibition of ASFV P30 protein synthesis in PAMs cells by different concentrations of toosendanin. The data were analyzed by one-way ANOVA followed by Dunnett's test.

[0030] Figure 5 Figure 5. Inhibition of PRRSV proliferation in PAMs and Marc-145 cells by different concentrations of toosendanin. The data were analyzed by one-way ANOVA followed by Dunnett's test.

[0031] Figure 6 Figure 6. Inhibition of PRRSV RNA proliferation in PAMs and Marc-145 cells by different concentrations of toosendanin. The data were analyzed by one-way ANOVA followed by Dunnett's test.

[0032] Figure 7 Figure 7. Inhibition of PRRSV N protein synthesis in PAMs and Marc-145 cells by different concentrations of toosendanin. The data were analyzed by one-way ANOVA followed by Dunnett's test.

[0033] Figure 8 Figure 8. Inhibition of PEDV proliferation in Vero cells by different concentrations of toosendanin. The data were analyzed by one-way ANOVA followed by Dunnett's test.

[0034] Figure 9 Figure 9. Inhibition of PEDV RNA proliferation in Vero cells by different concentrations of toosendanin. The data were analyzed by one-way ANOVA followed by Dunnett's test.

[0035] Figure 10 Figure 10. Inhibition of PEDV N protein synthesis in Vero cells by different concentrations of toosendanin. The data were analyzed by one-way ANOVA followed by Dunnett's test.

[0036] In the figures, "*" indicates significant difference (P < 0.05), "**" indicates extremely significant difference (P < 0.01), and "***" indicates extremely significant difference (P < 0.001) compared with the virus infection control group. DETAILED DESCRIPTION

[0037] The present application is further illustrated by the following description with reference to the accompanying drawings and specific examples. Unless otherwise specified, the reagents, methods and apparatus employed in the present application are of a kind commonly used in the art.

[0038] Wherein, the meliatoxin used in the present application is prepared by the following method:

[0039] The root bark of Melia toosendan was crushed by a pulverizer and sieved (20 mesh). The root bark was extracted twice by stirring in a 50℃ water bath with 90% ethanol solution for 2 hours each time. The filtrates from the two extractions were combined and distilled under reduced pressure to obtain a crude extract of Melia toosendan. A certain amount of the crude extract was weighed and extracted with petroleum ether, ethyl acetate and n-butanol in sequence. The ethyl acetate extract was distilled under reduced pressure to obtain a concentrated paste, which was then subjected to silica gel column chromatography. Elution was performed in sequence with 1% methanol-chloroform, 2% methanol-chloroform and 5% methanol-chloroform. The eluate containing meliatoxin was collected, concentrated under reduced pressure and then subjected to silica gel column chromatography again using 1% methanol-chloroform as the eluent. The eluate was then subjected to a Sephadex LH-20 column and eluted with 50% methanol-water to obtain purified meliatoxin. The purity of the purified meliatoxin was determined by HPLC to be 98.5%.

[0040] Unless otherwise specified, the reagents and materials used in the following examples were commercially available.

[0041] Example 1 Inhibitory effect of meliatoxin at different concentrations on the proliferation of ASFV DNA in PAMs cells

[0042] 1. Experimental method

[0043] Porcine alveolar macrophages (PAMs) were recovered (isolated from the lungs of slaughtered pigs and previously stored in a liquid nitrogen tank for use) and resuspended in RPMI-1640 culture medium (containing 10% fetal bovine serum, 100 U / mL penicillin and 100 U / mL streptomycin) and inoculated into a 24-well plate at a concentration of 8 x 10 5 6 cells / well. Meliatoxin was set at high (3 μM), medium (1 μM) and low concentrations (0.3 μM), a virus group (Mock), and a cell control group (NC, not infected, no drug). Each group had 3 replicates. The cells were incubated in a 37℃, 5% CO2 incubator for 6 hours. PAMs cells in the 24-well plate were infected with ASFV at a multiplicity of infection (MOI) of 2. After 90 minutes of infection, the supernatant virus solution was removed and 3 μM, 1 μM and 0.3 μM meliatoxin diluted in maintenance medium (RPMI-1640 culture medium containing 2% fetal bovine serum) was added. The cells were incubated in a 37℃, 5% CO2 incubator for 24 hours and then the culture was terminated.

[0044] After observing the cell morphology, the cell plate was repeatedly frozen and thawed at -80℃ and 4℃ for three times to fully lyse the cells so that the virus in the cells was all released into the cell supernatant, and then the supernatant of each well was collected. The collected cell supernatant was subjected to DNA extraction using the operation method recommended by a DNA extraction kit (Nanjing Nuowezan Biotechnology Co., Ltd.). The extracted DNA was used as a template, the ASFV p72 gene site was used as a target for identification, the ASFV UPLqPCR method recommended by the International Office of Epizootics (OIE) was used for detection, and the CT value of each treatment group was obtained. The p72 is an important structural protein of the ASFV and can reflect the number of viruses. By analyzing the CT value change of the p72 gene of the viruses treated in different manners, the inhibitory effect of the nitidine on the proliferation of the ASFV DNA in the PAMs was evaluated.

[0045] The upstream and downstream primer sequences of the ASFV p72 gene are as follows:

[0046] p72-F: 5'-CCCAGGRGATAAAATGACTG-3';

[0047] p72-R: 5'-CACTRGTTCCCTCCACCGATA-3'.

[0048] 2, Experimental results

[0049] The results are shown in Figure 1 As shown in the figure, the drug nitidine of the application has a significant inhibitory effect on the DNA proliferation of the ASFV in the PAMs in the concentration range of 0.3-3 μM, and shows a good dose-effect relationship.

[0050] Example 2: Inhibitory effect of nitidine with different concentrations on the RNA transcription of the ASFV in PAMs

[0051] 1, Experimental method:

[0052] The PAMs were resuspended by using the RPMI-1640 culture solution, and were inoculated into a 24-well plate at 8×10 5 cells / well; nitidine high (3 μM), medium (1 μM) and low concentration groups (0.3 μM), virus group (Mock), cell control group (NC) were set, each group had 3 repeats; 6 h in a 37℃, 5% CO2 cell incubator; the PAMs cell monolayer in the 24-well plate with a filling degree of about 80-90% was infected with 2 MOI of the ASFV, 90 min after the infection, the supernatant virus solution was removed, 3 μM, 1 μM and 0.3 μM concentrations of nitidine diluted and prepared by the maintenance solution (RPMI-1640 culture solution containing 2% fetal bovine serum) were added, and the incubation was continued for 24 h in a 37℃, 5% CO2 incubator, and then the culture was terminated.

[0053] After observing the cell morphology, the cell plate was repeatedly frozen and thawed at -80℃ and 4℃ for three times to fully lyse the cells, so that the virus in the cells was released into the cell supernatant, and then the supernatant of each well was collected. The collected cell supernatant was subjected to total RNA extraction by using the total RNA rapid extraction kit (Shanghai Feijie Biotechnology Co., Ltd.) recommended operation method. After the RNA was extracted, reverse transcription was immediately performed, and the copy number of the p30 gene of ASFV was detected by Real Time PCR with cDNA as a template and GAPDH as an internal reference gene; the virus control group was used as a reference to evaluate the change of p30 mRNA.

[0054] The upstream and downstream primer sequences of the PRRSV p30 gene are as follows:

[0055] p30-F: 5'-TGCACATCCTCCTTTGAAACAT-3';

[0056] p30-R: 5'-TCTTTTGTGCAAGCATATACAGCTT-3'.

[0057] The upstream and downstream primer sequences of the GAPDH gene are as follows:

[0058] GAPDH-F: 5'-GTCAGTGGTGGACCTGACCT-3';

[0059] GAPDH-R: 5'-TGCTGTAGCCAAATTCGTTG-3'.

[0060] 2. Experimental results:

[0061] The results are shown in Table 1. Figure 2 The drug toosendanin has a significant inhibitory effect on the RNA transcription of ASFV in PAMs in the concentration range of 0.3-3 μM, and shows a good dose-effect relationship.

[0062] Example 3: Western Blot analysis of the inhibitory effect of toosendanin with different concentrations on the synthesis of ASFV p30 protein in PAMs

[0063] 1. Experimental method:

[0064] The PAMs were resuscitated, resuspended with RPMI-1640 culture solution, and then 8×10 5Cells were seeded per well in 24-well plates; control group, virus group, and high (3 μM), medium (1 μM), and low (0.3 μM) concentration groups of azadirachtin were set up, with 3 replicates for each group; cells were cultured at 37°C and 5% CO2 for 6 h; PAMs cell monolayers with a filling degree of about 80-90% in 24-well plates were infected with 2 MOI of ASFV. After 90 min of infection, the supernatant virus solution was aspirated, and azadirachtin at concentrations of 3 μM, 1 μM, and 0.3 μM, prepared by diluting the maintenance medium (RPMI-1640 culture medium containing 2% fetal bovine serum), was added. The cells were then incubated at 37°C and 5% CO2 for another 24 h before the culture was terminated.

[0065] Discard the supernatant and wash twice with PBS. Place the cell culture plate on ice, add 45 μL of RIPA lysis buffer per well, repeatedly pipette and transfer the liquid to a centrifuge tube, centrifuge at 12,000 rpm for 10 min, and transfer the supernatant to another clean tube for later use. After determining the protein concentration of each sample using the BCA method, Western blotting was used to detect the bands of ASFV p30 protein and the internal reference protein α-Tubulin.

[0066] 2. Experimental Results:

[0067] See results Figure 3 As shown in the figure, the drug azadirachtin of the present invention has a significant inhibitory effect on the protein synthesis of ASFV in PAMs in the concentration range of 0.3 to 3 μM, and exhibits a good dose-response relationship.

[0068] Example 4: Immunofluorescence analysis of the inhibitory effect of different concentrations of azadirachtin on ASFV P30 protein synthesis in PAMs cells.

[0069] 1. Experimental Method:

[0070] Resuscitate PAMs, resuspend cells in RPMI-1640 medium, and administer at 8 × 10⁻⁶ ppm. 5 Cells were seeded per well in 24-well plates; control group, virus group, and high (3 μM), medium (1 μM), and low (0.3 μM) concentration groups of azadirachtin were set up, with 3 replicates for each group; cells were cultured at 37°C and 5% CO2 for 6 h; PAMs cell monolayers with a filling degree of about 80-90% in 24-well plates were infected with 2 MOI of ASFV. After 90 min of infection, the supernatant virus solution was aspirated, and azadirachtin at concentrations of 3 μM, 1 μM, and 0.3 μM, prepared by diluting the maintenance medium (RPMI-1640 culture medium containing 2% fetal bovine serum), was added. The cells were then incubated at 37°C and 5% CO2 for another 24 h before the culture was terminated.

[0071] Discard the supernatant, wash with PBS for 3 times, add 400 μL of 4% paraformaldehyde to fix for 30 min; wash with PBS for 3 times, add 200 μL of 0.25% Triton X-100 to permeabilize, and incubate at room temperature for 30 min; add 5% BSA to block the impurities, and incubate at room temperature for 1 h; wash with PBS for 3 times, add mouse p30 monoclonal antibody (diluted 1:5000), and incubate at 4°C overnight; wash with PBS for 3 times, add secondary antibody (Alexa 488-labeled anti-mouse IgG, 1:1000) in the dark, incubate at 37°C for 1 h, and wash with PBS; observe under a fluorescence microscope, and green fluorescence represents the p30 protein of ASFV.

[0072] 2. Experimental results:

[0073] Results are shown in Figure 4 As shown in the figure, the drug toosendanin of the present application has a significant inhibitory effect on the synthesis of p30 protein of ASFV in PAMs in a concentration range of 0.3-3 μM, and shows a good dose-effect relationship.

[0074] Example 5 Inhibitory effect of toosendanin with different concentrations on PRRSV proliferation in PAMs and Marc-145 cells

[0075] 1. Experimental method:

[0076] After PAMs and Marc-145 cells grow to a monolayer in a 6-hole cell culture plate, discard the culture medium, wash with PBS for 2 times, add 2 mL of PRRSV with 100 TCID 50 diluted in DMEM cell maintenance liquid containing 2% fetal bovine serum, and incubate at 37°C for 2 h; discard the virus supernatant, wash with PBS for 2 times, and add 2 mL of toosendanin with concentrations of 0.1, 0.4 and 1.6 μM, respectively. A normal control group (without the tested drug and PRRSV) and a PRRSV control group (without the tested drug) are set in the experiment, and toosendanin with different concentrations is used as an experimental group, and three parallel samples are set for each tested concentration. Continue to culture the cells at 37°C until the culture is terminated at 48 h after infection.

[0077] After observing the cytopathic effects, the cell plates were repeatedly freeze-thawed three times at -80℃ and 4℃ to ensure complete cell lysis, releasing all the virus into the cell supernatant. The supernatant from each well was then collected. The freeze-thawed samples were placed on ice and serially diluted 10-fold eight times with DMEM containing 2% fetal bovine serum. Marc-145 cells were removed and washed with PBS. The diluted samples were inoculated at 100 μL / well, one column per dilution. Each group was inoculated with virus-free maintenance medium as a negative control, and cultured further. After 2 hours, the cells were washed twice with PBS, and the blank maintenance medium was replaced. Cultured further. After 72 hours, the cytopathic effect was recorded, and the viral titer (TCID) was calculated. 50 value).

[0078] 2. Experimental Results:

[0079] See results Figure 5 As shown in the figure, the drug azadirachtin of the present invention has a significant inhibitory effect on PRRSV proliferation in Marc-145 cells in the concentration range of 0.1 to 1.6 μg / mL, and exhibits a good dose-response relationship.

[0080] Example 6: Inhibitory effect of different concentrations of azadirachtin on PRRSV RNA proliferation in PAMs and Marc-145 cells

[0081] 1. Experimental Method:

[0082] After PAMs and Marc-145 cells have grown to a monolayer in 6-well cell culture plates, the culture medium is discarded, the cells are washed twice with PBS, and then diluted with DMEM cell maintenance medium containing 2% fetal bovine serum and 100 TCID50 is added. 50 PRRSV was incubated at 2 mL / well at 37°C for 2 h. The viral supernatant was discarded, and the cells were washed twice with PBS. Then, 0.1, 0.4, and 1.6 μM azadirachtin were added at 2 mL / well, respectively. A normal control group and a PRRSV control group were included in the experiment. Different concentrations of azadirachtin were used as experimental groups, with three replicates for each tested concentration. Cells were cultured at 37°C for 48 h post-infection before termination of culture.

[0083] After observing the lesion condition, the cell plates were repeatedly frozen and thawed three times at -80℃ and 4℃ to ensure complete cell lysis, resulting in the release of all intracellular viruses into the cell supernatant. The supernatant from each well was then collected. Total RNA was extracted from the collected cell supernatant using the recommended procedure of the Total RNA Rapid Extraction Kit (Shanghai Feijie Biotechnology Co., Ltd.). Immediately after RNA extraction, reverse transcription was performed. Using cDNA as a template and GAPDH as an internal reference gene, Real-Time PCR was used to detect the copy number of the PRRSV NSP9 gene. A normal control group was used as a reference to evaluate changes in NSP9 mRNA.

[0084] PRRSV NSP9 gene up and down stream primer sequences:

[0085] NSP9-F: 5'-CTAAGAGAGGTGGCCTGTCG-3';

[0086] NSP9-R: 5'-GAGACTCGGCATACAGCACA-3'.

[0087] GAPDH gene up and down stream primer sequences:

[0088] GAPDH-F: 5'-GTCAGTGGTGGACCTGACCT-3';

[0089] GAPDH-R: 5'-TGCTGTAGCCAAATTCGTTG-3'.

[0090] 2. Experimental results:

[0091] Results are shown in Figure 6 As shown in the figure, the drug toosendanin of the application has a significant inhibitory effect on the proliferation of PRRSV RNA in Marc-145 cells in the concentration range of 0.1-1.6 μM, and shows a good dose-effect relationship.

[0092] Example 7: Western Blot analysis of the inhibitory effect of toosendanin at different concentrations on the synthesis of PRRSV N protein in PAMs and Marc-145 cells

[0093] 1. Experimental method:

[0094] After the PAMs and Marc-145 cells grow to a monolayer in the 6-well cell culture plate, the culture medium is discarded, and the cells are washed with PBS for 2 times, 2 mL of PRRSV containing 100 TCID 50 / mL diluted with DMEM cell maintenance solution containing 2% fetal bovine serum is added, 2 mL / well, 37°C incubation for 2 h, the virus supernatant is discarded, and the cells are washed with PBS for 2 times, 0.1, 0.4, and 1.6 μM of toosendanin is added, 2 mL / well. The normal control group and the PRRSV control group are set up at the same time, and different concentrations of toosendanin are used as experimental groups, and three parallel tests are set up for each test concentration. The cells are continuously cultured at 37°C until 48 h after infection.

[0095] The supernatant is discarded, and the cells are washed with PBS for 2 times. The cell culture plate is placed on ice, 150 μL of RIPA lysis buffer is added per well, the liquid is blown out into a centrifuge tube after blowing, and centrifugation is performed at 15000 rpm for 15 minutes, and the supernatant is sucked out into another clean tube for standby. After determining the protein concentration of each sample by BCA method, the bands of PRRSV N protein and internal reference protein GAPDH are detected by Western Blot.

[0096] 2. Experimental Results:

[0097] See results Figure 7 As shown in the figure, the drug azadirachtin of the present invention has a significant inhibitory effect on the protein synthesis of PRRSV in PAMs and Marc-145 cells in the concentration range of 0.1 to 1.6 μM, and exhibits a good dose-response relationship.

[0098] Example 8: Inhibitory effect of different concentrations of azadirachtin on PEDV proliferation in Vero cells

[0099] 1. Experimental Method:

[0100] With a density of 1.5 × 10 5 Vero cells were seeded at a density of 100 cells / mL in 96-well plates. After the cells reached confluence and formed a monolayer, they were washed twice with PBS. The compound was serially diluted twofold with DMEM maintenance medium containing 2% FBS. Six concentration gradients of TSN drug (0.5-16 μM) were set up, along with a solvent control group containing 4‰ DMSO and a blank control group, with 100 μL per well. The cells were incubated at 37°C and 5% CO2 for 48 h before culture was terminated. After observing cell morphology, the cells were fixed with 4% paraformaldehyde for IFA detection, and observed and photographed using a fluorescence inverted microscope. The fluorescence intensity (blue and red fluorescence) of each well was quantified using ImageJ software. The DMSO-treated control group was set as 100%, and the other groups were compared with the DMSO-treated group. The half-maximal effective concentration (EC50) was determined by the quantified cell protection rate of the drug treatment group. 50 The values ​​were calculated using a nonlinear regression function with GraphPad Prism 8.0 software.

[0101] The calculation formula is as follows:

[0102]

[0103] 2. Experimental Results:

[0104] See results Figure 8 As shown in the figure, the drug azadirachtin of the present invention has antiviral activity against PEDV virus, and its EC50... 50 The value is 1.54 μM.

[0105] Example 9: Inhibitory effect of different concentrations of azadirachtin on PEDV RNA proliferation in Vero cells

[0106] 1. Experimental Method:

[0107] After the Vero cells are grown to a monolayer in a 6-well cell culture, they are washed twice with PBS, 2 mL of PEDV containing 100 TCID 50 diluted in DMEM cell maintenance liquid containing 2% fetal bovine serum is added, 2 mL / well, 37°C incubation for 2 h, the virus supernatant is discarded, and the cells are washed twice with PBS, and 2, 4 and 8 μM of the morinda citrifolia extract is added, 2 mL / well. The experiment also includes a normal control group, a PEDV control group, and different concentrations of the morinda citrifolia extract as experimental groups, and three parallel tests are performed for each test concentration. The cells are continuously cultured at 37°C until the culture is terminated at 48 h after infection.

[0108] After observing the cytopathic effect, the cell plates are repeatedly frozen and thawed at -80°C and 4°C for three times to fully lyse the cells, so that all the viruses in the cells are released into the cell supernatant, and then the supernatant of each well is collected. The collected cell supernatant is subjected to total RNA extraction by using the total RNA rapid extraction kit (Shanghai Feijie Biotechnology Co., Ltd.) according to the recommended operation method. After the RNA is extracted, reverse transcription is immediately performed, cDNA is used as a template, β-Actin is used as an internal reference gene, Real Time PCR is used to detect the copy number of the PEDV N gene; the normal control group is used as a reference to evaluate the change of the virus N protein mRNA.

[0109] The upstream and downstream primer sequences of the PEDV N gene are as follows:

[0110] PEDV N-F: 5'-CGCAAAGACTGAACCCACTAATTT-3';

[0111] PEDV N-R: 5'-TTGCCTCTGTTGTTACTTGGAGAT-3'.

[0112] The upstream and downstream primer sequences of the β-Actin gene are as follows:

[0113] β-Actin-F: 5'-GGACTTCGAGCAGGAGATGG-3';

[0114] β-Actin-R: 5'-AGGAAGGAGGGCTGGAAGAG-3'.

[0115] 2. Experimental results:

[0116] The results are shown in Figure 9 As shown in the figure, the morinda citrifolia extract of the application has a significant inhibitory effect on the proliferation of PEDV RNA in the Vero cells in the concentration range of 2-8 μM, and shows a good dose-effect relationship.

[0117] Example 10: Analysis of the inhibitory effect of different concentrations of the morinda citrifolia extract on the synthesis of PEDV N protein in Vero cells by immunofluorescence method

[0118] 1. Experimental Method:

[0119] Vero cells were seeded into 96-well plates and allowed to grow into a confluent monolayer. The cells were washed twice with PBS, and the virus was diluted to 100 TCID using DMEM maintenance medium containing 2% FBS. 50 Viral fluid was prepared, and a blank control group was set up. After incubation at 37℃ and 5% CO2 for 2 hours, the cells were washed twice with PBS to remove unbound viral particles. A blank control group, a solvent control group, and groups containing different concentrations of TSN were also prepared, with 100 μL per well. After 48 hours of incubation, the supernatant was discarded, and the cells were washed three times with PBS. 150 μL of 4% paraformaldehyde was added to each well for fixation for 30 min. After three washes with PBS, 50 μL of 0.25% Triton X-100 was added for permeabilization, and the cells were incubated at room temperature for 10 min. After three washes with PBS, 50 μL of 5% BSA was added to block contaminating proteins, and the cells were blocked at room temperature for 1 hour. After three washes with PBS, 50 μL of mouse-derived PEDV N protein monoclonal antibody (1:800 dilution) was added, and the cells were incubated overnight at 4℃. After three washes with PBS, 50 μL of secondary antibody (Alexa) was added in the dark. 568-labeled goat anti-mouse IgG (H+L, 1:1000) was incubated at 37°C in the dark. After 1 hour, the cells were washed three times with PBS for 5 minutes each time. 50 μL of DAPI (300 nM) solution was added to each well in the dark, and the cells were incubated at room temperature for 5 minutes. The cells were then washed three times with PBS for 5 minutes each time. Observation and photographing were performed using an inverted fluorescence microscope. Red fluorescence represents the N protein of PEDV, and blue fluorescence represents the Vero cell nucleus.

[0120] 2. Experimental Results:

[0121] See results Figure 10 As shown in the figure, the drug azadirachtin of the present invention, within a concentration range of 2-8 μM, significantly inhibited the synthesis of N protein of PEDV virus in Vero cells after 48 h of treatment, and exhibited a good dose-response relationship.

[0122] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. The use of toosendanin in the preparation of a medicine for preventing and treating porcine reproductive and respiratory syndrome, characterized in that, The pig reproductive and respiratory syndrome is caused by pig reproductive and respiratory syndrome virus infection.

2. Use according to claim 1, characterized in that, The effective concentration range of the xiaofengtong for inhibiting the proliferation of the pig reproductive and respiratory syndrome virus is (0.1-1.6) μM.

3. Use according to any one of claims 1 to 2, characterized in that, The xiaofengtong is extracted from the root bark and / or bark of Melia toosendan or from the fruit of Melia toosendan.

4. Use according to claim 3, characterized in that, The xiaofengtong is prepared by the following method: The root bark and / or bark of Melia toosendan is crushed and sieved, and 80-100% ethanol solution is heated in a water bath at 45-60 ℃ for 1-3 hours for 1-3 times, the filtrates are combined, and concentrated to obtain a crude extract; the crude extract is extracted with petroleum ether, ethyl acetate and n-butanol in sequence, and the ethyl acetate extraction part is concentrated under reduced pressure; the obtained concentrated paste is subjected to silica gel column chromatography, eluted with 1% methanol-chloroform, 2% methanol-chloroform and 5% methanol-chloroform in sequence, the eluate containing xiaofengtong is collected, concentrated under reduced pressure, and subjected to silica gel column chromatography again and eluted with 1% methanol-chloroform; the eluate is subjected to dextran gel LH-20 column and eluted with 50% methanol-water to obtain purified xiaofengtong.

5. Use according to claim 1, characterized in that, The medicine is an injection preparation, an oral preparation, an atomization inhalation preparation or a transdermal preparation.

Images